1. Introduction
2. Background of the Invention
2.1. Diltiazem and its Analogues
2.2. The Stereochemistry of Diltiazem and Its Precursors
2.3. Techniques for Resolution of Glycidate Esters
2.4. Enzymatic Resolution of Racemic Mixtures
3. Summary of the Invention
4. Brief Description of the Figures
5. Detailed Description of the Invention
5.1. Multiphase Enzymatic Reaction Processes
5.2. Examples of Multiphase Enzymatic Resolutions
5.2.1. Procedures for Examples 1-6
5.2.2. Example 1xe2x80x94Stereoselective Hydrolysis of trans-3-(4-methoxy-phenyl)glycidic Acid Methyl Ester in tert-butyl Methyl Ether
5.2.3. Example 2xe2x80x94Stereoselective Hydrolysis of trans-3-(4-methoxy-phenyl)glycidic Acid Methyl Ester in Toluene
5.2.4. Example 3xe2x80x94Stereoselective Hydrolysis of trans-3-(4-methoxy-phenyl)glycidic Acid Ethyl Ester in tert-butyl Methyl Ether
5.2.5. Example 4xe2x80x94Stereoselective Hydrolysis of trans-3-(4-methoxy-phenyl)glycidic Acid n-Butyl Ester in tert-Butyl Methyl Ether
5.2.6. Example 5xe2x80x94Stereoselective Hydrolysis of trans-3-(4-methoxy-phenyl)glycidic Acid Isopropyl Ester in tert-Butyl Methyl Ether
5.2.7. Example 6xe2x80x94Stereoseletive Hydrolysis of trans-3-(4-methoxy-phenyl)glycidic Acid Isobutyl Ester in tert-Butyl Methyl Ether
5.2.8. Effect of Cosolvent on Apparent Enantioselectivity E: Example 7
5.2.9. Effect of Water-Immiscible Organic Solvent on Apparent Enatio-selectivity E: Example 8
5.2.10. Effect of pH on Enzyme Enatio-selectivity E: Example 9
5.2.11. Methyl Ester Hydrolysis Catalyzed by Lipase MAPxe2x80x94Examples 10 and 11
5.2.12. Methyl Ester Hydrolysis Catalyzed by Lipase-OFxe2x80x94Example 12
5.2.13. Resolution of Racemic Methyl 3-(4-Methoxyphenyl)-glycidate in a Membrane Reactor at pH 7xe2x80x94Example 13
5.2.14. Resolution of Racemic Methyl 3-(4-Methoxyphenyl)-glycidate in a Membrane Reactor at pH 8xe2x80x94Examples 14-17
5.3. Management of the Aldehyde Byproduct by Adduct Formation with Bisulfite
5.4. Examples Pertinent of Bisulfite Utilization
5.4.1. Resolution of trans-3-(4-Methoxy-phenyl)glycidic Acid Methyl Ester in a Multiphase Enzyme Membrane Reactor
5.4.2. Example 18xe2x80x94resolution of trans-3-(4-Methoxyphenyl)glycidic Acid Methyl Ester in a Multiphase Enzyme Membrane Reactor in the Absence of Bisulfite
5.4.3. Example 19xe2x80x94Resolution of trans-3-(4-Methoxyphenyl)glycidic Acid Methyl Ester in a Multiphase Enzyme Membrane Reactor Using Sodium Bisulfite
5.4.4. Example 20xe2x80x94Enrichment of Reaction Product from Enzymatic Resolution of trans-3-(4-Methoxyphenyl)glycidic Acid Methyl Ester Using Sodium Bisulfite
5.4.5. Example 21xe2x80x94Recovery of trans-3-(4-Methoxyphenyl)glycidic Acid Methyl Ester from Tolune Using Concentrated Sodium Bisulfite
5.4.6. Example 22xe2x80x94Degree of Inhibition on Representative Enzymes of Reaction Products
5.4.7. Example 23xe2x80x94Resolution of trans-3-(4-Methoxyphenyl)glycidic Acid Methyl Ester Using Sodium Bisulfite
5.4.8. Example 24xe2x80x94Resolution of trans-3-(4-Methoxyphenyl)glycidic Acid Methyl Ester in the Absence of Sodium Bisulfite
5.4.9. Example 25xe2x80x94Resolution of trans-3-(4-Methoxyphenyl)glycidic Acid Methyl Ester Using Sodium Bisulfite and Extended Reaction Time
5.4.10. Example 26xe2x80x94Resolution of trans-3-(4-Methoxyphenyl)glycidic Acid Methyl Ester Using Sodium Bisulfite in a Larger Scale
5.4.11. Example 27xe2x80x94Resolution of trans-3-(4-Methoxyphenyl)glycidic Acid Methyl Ester in a Multiphase Enzyme Membrane Reactor Using Palatase M
5.4.12. Example 28xe2x80x94Batch Reactions in Toluene Followed by Direct Crystallization of trans-3-(4-methoxyphenyl glycidic Acid Ester (GLOP) Therefrom
5.4.13. Example 29xe2x80x94Bench-Scale Dispersed Phase Enzymatic Resolution Providing High Optical Purity Solutions
5.4.14. Example 30xe2x80x94Large-Scale Membrane Reactor Process Providing Organic Solutions of Highly Resolved GLOP
5.4.15. Stability of GLOP in Some Representative Types of Solvents
5.5. Further Considerations with Regard to Choice of Solvent
5.5.1. The Effect of Solvent on Enzyme Activity
5.5.2. Determination of the Non-Enzymatic Degradation of Substrate Ester
5.6. Post-Enzyme Resolution Clean-Up of Crude Reaction Products by Extraction with a Carbonyl Adduct-Forming Agent
5.6.1. Example 34xe2x80x94Purification of Crude Organic Solution by Extraction with Aqueous Bisulfite Anion
5.7. Reactivity of the Oxirane Ring
The esters of trans-3-(4-methoxyphenyl)glycidic acid have utility as precursors in the chemical synthesis of diltiazem. Moreover, these compounds present a very attractive point in the overall synthetic route to diltiazem at which to introduce the desired stereochemistry into the diltiazem precursors through resolution of the racemic glycidic esters and use of the correct, optically purified precursor ester. The present invention pertains to a novel enzymatic method for resolving a racemic mixture of esters of the (2R,3S)- and (2S,3R)-enantiomers of trans-3-(4-methoxyphenyl)glycidic acid. It also pertains to a process for diltiazem production incorporating this resolution step and to membrane reactor means for improving the efficiency of enzymatic resolution of this diltiazem intermediate. The amelioration of the effects of an inhibitory aldehyde by-product of the reaction process by means of its formation of an adduct with an agent provided in the aqueous reaction phase is also an aspect of the present invention. Moreover, selected organic solutions of the optically active glycidic ester intermediates have been developed which are particularly useful in subsequent processes involving the isolation of the intermediate directly from the solution or use of the solution to introduce a new reagent for a further chemical transformation.
Diltiazem, the chemical structure of which is shown in FIG. 1, is an optically active pharmaceutical compound. More specifically, diltiazem, the chemical name of which is (+)-5-[2-(dimethylamino)ethyl]-cis-2,3-dihydro-3-hydroxy-2-(p-methoxyphenyl)-1,5-benzothiazapin-4(5)-one acetate (ester), consists of a substituted benzothiazapene wherein both chiral carbon atoms have the S absolute stereo-configuration (H. Inoue et al., U.S. Pat. No. 3,562,257). Diltiazem has proven useful for the treatment of angina due to coronary artery spasm, and for exertional angina. The beneficial therapeutic effects achieved with diltiazem are believed to be derived from the inhibition of calcium ion influx during depolarization of the cell membrane in both cardiac and smooth muscle. Diltiazem is known to prevent coronary artery spasm, both spontaneous and ergonovine provoked, and to decrease peripheral vascular resistance. Diltiazem is marketed by Tanabe and by Marion Laboratories in the United States, where it is sold under the tradename Cardizem(copyright). Analogues to diltiazem are also known to exist, e.g., wherein the benzothiazapene moiety has a single chlorine substituent on the aromatic ring.
Diltiazem is currently being manufactured via a process similar to that shown in FIG. 2. The first step in the synthetic sequence involves the Lewis acid-catalyzed nucleophilic attack of o-nitrothiophenol on methyl trans-3-(4-methoxyphenyl)glycidate, as a mixture of enantiomers, to give the threo compound shown (H. Inoue et al., J. Chem. Soc. Perkin Trans. I, 1984, 1725; H. Inoue et al., J. Chem. Soc. Perkin Trans. I, 1985, 421; H. Inoue et al., U.S. Pat. No. 4,420,628). This threo compound then needs to be resolved at a subsequent step in the synthetic pathway in order to arrive at the optically active final product (diltiazem).
Alternative production routes to diltiazem utilize o-aminothiophenol in place of o-nitrothiophenol in the step involving opening of and addition to the oxirane ring (S. Nagao et al., U.S. Pat. No. 4,416,819). Such alternative processes also utilize methyl trans-3-(4-methoxyphenyl)-glycidate as an intermediate, and thus are subject to improvement by the process of the present invention.
It is known that the above pharmacological effects reside in only one of the two enantiomers of the diltiazem, namely, the d-enantiomer (Merck Index, 10th Edition, 1986, p. 466; Physician""s Desk Reference, 41st Edition, 1987, p. 1173). Thus, there is a need to produce diltiazem with the correct stereochemistry, and to introduce such correct stereochemistry at an efficient point in the overall synthesis by production of an optically purified and stereochemically correct intermediate or precursor. As discussed above, diltiazem can be produced from a 3-(4-methoxyphenyl)glycidic acid ester intermediate. At the present time, this intermediate is used in its racemic form, i.e., it is not optically active.
The 3-(4-methoxyphenyl)glycidic acid ester, shown in FIG. 3, contains two chiral centers at carbon atoms 2 and 3, both of which may assume either the R or S absolute configurations. Generally speaking, molecules containing n chiral centers and having no elements of reflective symmetry, will have 2n stereoisomers. In a molecule with 2 chiral centers, there will thus be 22 or 4 stereoisomers. Furthermore, in the case of a molecule having only two chiral centers, these four stereoisomers will be related as a diastereomeric pair of enantiomers, that is two diastereomers each existing as a mixture of its two enantiomers. In the specific case of 3-(4-methoxyphenyl)glycidic acid esters, the two diastereomeric forms are described as cis and trans isomers. The cis isomer is defined as the diastereomer in which the two hydrogen atoms bonded to the carbon atoms of the oxirane ring, that is carbon atoms 2 and 3, eclipse each other, that is, are on the same side of the plane defined by the oxirane ring substructure of the molecule. The trans isomer is defined as the diastereomer in which the hydrogen atoms bonded to carbon atoms 2 and 3 lie on opposite sides of the plane of the oxirane ring. Thus the relative configurations of carbon atoms 2 and 3 are fixed in each diastereomer, although each diastereomer will still exist as a pair of enantiomers. Because diastereomeric compounds are physically distinct entities, not related by symmetry operations performed on the entire molecule, they are physically distinguishable and may be produced separately by the appropriate conventional chemical methods.
The thermodynamically favorable trans diastereomer of a given 3-(4-methoxyphenyl)glycidic acid ester can be synthesized via the Darzen""s glycidic ester condensation, and rendered free of any cis diastereomer by conventional purification methods. The trans diastereomer exists in two enantiomeric forms, one having absolute configuration R at carbon atom 2, and absolute configuration S at carbon atom 3. This enantiomer is described as the (2R,3S) isomer. The other enantiomer of the trans diastereomer will have absolute configuration (2S,3R). The enantiomers of the cis diastereomer exhibit absolute configurations (2S,3S) and (2R,3R). The particular glycidic ester enantiomer having absolute configuration (2R,3S) is the compound desired as an optically purified synthetic precursor to diltiazem.
The production of (2R,3S)-3-(4-methoxyphenyl)glycidic acid methyl ester has previously been achieved through two fundamentally different procedures. The first procedure involves the synthesis of the chiral glycidic acid methyl ester from achiral precursors, with the creation of chirality during a specific reaction which utilizes a chiral oxidation reagent. Thus, trans-cinnamyl alcohol is asymmetrically epoxidized to give the desired oxirane ring structure, with the correct stereochemistry being created at carbon atoms 2 and 3 simultaneously (K. Igarashi et al., U.S. Pat No. 4,552,695).
The second procedure, which is generally considered more classical, involves the use of an optically pure reagent used in stoichiometric quantities, to form diastereomeric adducts with the enantiomers of racemic esters or salts of trans-3-(4-methoxyphenyl)glycidic acid (M. Hayashi et al., Japan 5 Kokai Tokkyo Koho J P 61/145160 A2 [86/145160] (1986); M. Hayashi et al., Japan Kokai Tokkyo Koho J P 61/145160 A2 [86/145160] (1986)). These adducts are physically distinguishable, and may be separated by conventional procedures such as fractional crystallization. The thus separated adducts are then decomposed under controlled conditions to leave the separated enantiomers, and the recovered resolving reagent.
Both of these procedures suffer drawbacks, however. In particular, the first procedure involves the use of an unusual catalyst, namely, dialkyl tartrate titanium(IV) isopropoxide, which requires anhydrous conditions and concomitant handling procedures (K. B. Sharpless et al., J. Amer. Chem. Soc., 1980, 102, 5974; K. B. Sharpless et al., Pure Appl. Chem., 1983, 55, 589). More importantly, the reaction which creates the desired stereochemistry does not produce the methyl ester directly. Two further reactions are required beyond the point at which chirality is introduced, involving the production (by oxidation of the alcohol) and esterification of the glycidic acid itself, which is an unstable compound requiring special handling. The second procedure suffers from the need for stoichiometric quantities of previously resolved chiral materials or resolving agents such as alpha-methylbenzylamine (S. Nagao et al., U.S. Pat. No. 4,416,819). Because of the expense of such resolving agents, there also exists a need to recover these materials in a quantitative manner after the resolution step. Additionally, the energy and solvent requirements of large-scale crystallization processes make them unattractive.
Another approach to the resolution of racemic mixtures of chiral compounds involves subjecting racemic compounds to the enantioselective action of various enzymes. Enzymatic resolution has been widely employed for the lab-scale, preparative-scale, and industrial-scale production of many optically pure compounds including esters but not heretofore the glycidate esters.
Many different classes of enzymes have been used for the resolution of stereoisomers on a preparative scale, including hydrolases (especially the lipases, proteases and esterases such as chymotrypsin), lyases and oxidoreductases (e.g., amino acid oxidases and alcohol reductases). Generally speaking, enzymes for use in resolutions should ideally exhibit broad substrate specificity, so that they will be capable of catalyzing reactions of a wide range of xe2x80x9cunnaturalxe2x80x9d substrates, and they should exhibit a high degree of stereoselectivity for catalyzing the reaction of one isomer to the exclusion of others.
The hydrolases (e.g., lipases, proteases and esterases) are among the more attractive enzymes for use in resolutions, because they are commercially available at reasonable cost, they do not require expensive cofactors, and some of them exhibit reasonable tolerance to organic solvents. Additionally, chiral chemistry often involves alcohols, carboxylic acids, esters, amides and amines with chiral carbons, and carboxyl hydrolases are preferred choices as stereoselective catalysts for reactions of such species (Cambou, B. and A. M. Klibanov, Biotechnol. Bioeng., 1984, 26, 1449). Many pharmaceuticals and their intermediates exhibit very low solubilities in water, and accordingly a number of enzyme-mediated optical resolutions have been conducted under multiphasic reaction conditions.
Enzymatic treatment has been applied to the resolution of racemic mixtures of amino acid esters. For example, Stauffer (U.S. Pat. No. 3,963,573) produced optically pure N-acyl-L-methionine by treating N-acyl-D,L-methionine ester with microbial proteases and separating the product acid from the reaction mixture. Similarly, Bauer (U.S. Pat. No. 4,262,092) prepared optically pure D-phenylalanine ester by subjecting a racemic mixture of an N-acyl-D,L-phenyl-alanine ester to the action of a serine protease, separating the unaffected N-acyl-D-phenylalanine ester, and removing the N-acyl and ester groups. Matta et al. (i J. Org. Chem., 1974, 39, 2291) used chymotrypsin in the resolution of precursors of the drug 3-(3,4-dihydroxyphenyl)alanine or dopa.
Enzymes have also been explored for the resolution of other compounds such as agricultural chemicals, sometimes in biphasic reactions systems. In particular, Cambou and Klibanov (Biotech. Bioeng., 1984, 26, 1449) examined the use of lipase immobilized in porous beads for the enzymatic resolution of mixtures of (R,S)-2-(p-chloro-phenoxy)propanoic acid (whose R isomer is a herbicide) and various esters thereof. The differing solubility properties of the acids and esters used in their studies required the dispersion and agitation of mixtures containing the immobilized solid-phase enzyme, an aqueous buffer, and the water-immiscible organic phase containing solvent and reactantxe2x80x94a relatively inefficient process.
Additional examples can be provided of the state-of-the-art of enzyme-mediated resolution as applied to the production of optically purified pharmaceuticals, albeit not to the enzymatic resolution of diltiazem precursors. Sih (U.S. Pat. No. 4,584,270) has disclosed enzymatic means for the production of optically pure (R)-4-amino-3-hydroxy-butanoic acid, a key intermediate in the preparation of L-carnitine. Additionally, certain optically pure D-amino acids (in particular, the D-arylglycines such as phenylglycine and 4-hydroxyphenylglycine) are used as side chains in the manufacture of semisynthetic penicillins and cephalosporins. Schutt et al. (Biotechnol. Bioeng., 1985, 27, 420) have subjected racemic mixtures of such nonpolar N-acyl-D,L-amino acid esters to the hydrolytic action of subtilisin in two-phase systems for the purpose of obtaining optically purified D-amino acids. In still other references, enzymes derived from microorganisms were utilized to resolve esters of naproxen and ibuprofen. C. J. Sih et al. (Tetrahedron Letters, 1986, 27, 1763) describes that esters of ibuprofen and naproxen are capable of being stereospecifically resolved using a microorganism-derived lipase.
In summary, there exists a need in the art for more efficient methods for production of optically purified diltiazem and its analogues, and in particular for improved processes for the optical resolution of racemic diltiazem precursors including the esters of trans-3-(4-methoxyphenyl) glycidic acid. Furthermore, while enzymatic resolution techniques have been employed for the production of many optically pure pharmaceuticals and their precursors, this technique has not yet been disclosed and successfully applied to the resolution of the glycidate esters that are chiral intermediates in the production of diltiazem. The present invention provides such an enzymatic resolution method.
The resolution process of the present invention is accomplished through the use of an enzyme that preferentially catalyzes hydrolysis of one of the two enantiomers of a given glycidic ester to the parent glycidic acid, leaving intact the enantiomer having the desired absolute configuration as the glycidic ester. A specific embodiment of this invention pertains to hydrolytic enzymes, with particularly preferred enzymes being chosen from the lipases and proteases, capable of preferentially hydrolyzing simple alkyl esters (e.g., the methyl ester) of (2S,3R)-methoxyphenylglycidate at a rate higher than the rate of hydrolysis of the corresponding (2R,3S)-enantiomer of methyl methoxyphenylglycidate, permitting recovery of the latter species in optically purified form for use as an optically resolved intermediate in the production of diltiazem.
Also included in this invention and described herein are methods for the efficient conduct of the enzymatic resolution step, including the use of multiphase and extractive membrane reactors to improve the efficiency of the biocatalytic reaction, as well as the provision of bisulfite anion in the aqueous reaction phase for the purpose of forming an adduct with an otherwise inhibitory and troublesome aldehyde by-product.
The present invention provides a method for obtaining an organic solution comprising an optically active diastereomer of a glycidic acid ester which involves preparing an organic solution comprising the diastereomer in a water-immiscible organic solvent, which diastereomer may be present as a mixture of enantiomers. This solution is then brought into contact with an aqueous mixture comprising water and an enzyme which catalyzes the enantioselective hydrolysis of the undesired glycidyl ester, thus enriching the organic solution in the desired enantiomer. The product enantiomer may then be crystallized directly from the enriched solution or may then be employed, while in this same solution, in a subsequent reaction.